One of the early applications of the trans-splicing phenomenon has been proposed by Sullenger and Cech (Nature, 1994, 371, 619-622) who described experiments in which ribozyme-mediated trans-splicing was used to replace a defective portion of RNA with a functional structure by using reengineered Tetrahymena group I intron to generate translatable lacZ transcripts in E. coli. They proposed trans-splicing as a general means of altering the sequence of specific host transcripts for the purposes of treatment of many genetic diseases. Another use of trans-splicing has been proposed by Ayre and colleagues, (1999, Proc. Natl. Acad. Sci. USA, 96, 3507-3512,), who developed a technology that utilizes ribozyme-mediated trans-splicing to target cytotoxins into cells in a highly specific manner. They used group I intron to splice the mRNA for Diphteria toxin A with virus mRNA to inactivate cells expressing viral mRNA, thus selectively inactivating infected yeast cells.
Yet another important application has been developed by Mikheeva and Jarrell (1996, Proc. Natl. Acad. Sci. USA, 93, 7486-7490) who used engineered group II introns to catalyze assembly of a chimeric gene. In this work, the ribozyme was modified so as to shuffle the mRNA of tissue plasminogen activator, and the resulting chimeric RNA was reverse transcribed into DNA. This approach allows to unidirectionally create libraries of genes that encode chimeric proteins with novel functions.
A general problem encountered in all of the above mentioned approaches is that cleaving ribozymes are unable to deplete living cells of the chosen target RNA. Further, the trans-splicing efficiency is very low leading to very low amounts of trans-spliced RNA in a cell. In many cells, no trans-spliced RNA product is obtained at all. Attempts have been made to use ribozymes with an extended complementarity to the target RNA and to design a precise alteration of the guide sequences required for substrate recognition (Kohler et al., 1999, J. Mol. Biol., 285, 1935-1950). Still, trans-splicing is inefficient and thus the applications of in vivo ribozyme-mediated trans-splicing today are limited to specific inactivation of cells using potent toxins trans-spliced with a highly abundant target RNA (of viral coat protein), a process that can effectively function even in the presence of unspliced target mRNA in a host cell.
It is therefore an object of the invention to provide an efficient process of amplification or expression of a sequence of interest in a cell.
It is an object of the invention to provide a biologically safe process of amplification or expression of a sequence of interest in a cell.
It is another object of the invention to provide an efficient process of assembling RNA, notably mRNA, of interest in a cell from precursor RNA molecules.
It is a further object to provide a process of assembling mRNA in a cell from a residential RNA and an externally provided RNA, whereby the effect of any remaining residential RNA is negligible compared to the assembled RNA.
It is a further object to provide a process of amplifying or down-regulating selected mRNA in a cell.
It is a further object to provide a method of increasing the efficiency of trans-splicing.